硫化氢对急性病毒性心肌炎小鼠心肌细胞凋亡的保护作用

Abstract

目的：探讨硫化氢(H2S)对CVB3诱导的急性病毒性心肌炎(Acute Viral Myocarditis, AVMC) Balb/c小鼠心肌细胞抗凋亡的保护作用。方法：将50只4周龄雄性Balb/c小鼠随机分为正常对照组(N组)、病毒感染组(C组)、硫化氢治疗组(H组)，C、H组小鼠腹腔反复接种0.2 mL内含柯萨奇病毒B3(CVB3)的1640培养液建立VMC模型，H组病毒感染同时腹腔注射50 μmol/kg NaHS，N组腹腔无菌注射等量生理盐水，1次/d，连续7天。动态观察记录实验期间各组小鼠一般情况，各组小鼠死亡数，绘制生存曲线进行生存分析；各组小鼠于第7天全部处死称重(BM)，取心脏称重(HM)，计算HM/BM；心肌HE染色后光镜下观察心肌病理改变并计算心肌病理积分；TUNEI法检测心肌细胞凋亡情况并计算心肌凋亡指数；Western Blot 法检测心肌细胞凋亡相关蛋白p53、Bax、Bcl-2、caspase-3的表达情况。结果：与N组相比，C组HM/BM比值增大、心肌病理积分较对照组明显增加，促凋亡蛋白p53、Bax表达水平增加，抑凋亡蛋白Bcl-2表达水平下降，caspase-3表达活性显著增加(p < 0.05)。在NaHS治疗后，H组与C组相比，H组的心肌病理积分明显降低，促凋亡蛋白p53、Bax表达水平明显下调，抑凋亡蛋白Bcl-2表达水平上调，caspase-3活性明显降低(p < 0.05)。结论：NaHS可提高VMC小鼠的存活率、降低心肌病理积分、减少心肌细胞凋亡数，并通过调节凋亡相关蛋白p53、Bax、Bcl-2、caspase-3表达抑制心肌细胞凋亡，从而起到保护VMC小鼠心肌的作用。 Objective: To explore the cardiomyocytes anti-apoptosis effect of Hydrogen Sulfide (H2S) on CVB3- induced Acute Viral Myocarditis (AVMC) Balb/c mice. Methods: 50 male 4 - 5-week-old Balb/c mice were randomly divided into three groups, including group N (control group, n = 10), group C (CVB3 virus control group, n = 20), and group H (NaHS treatment group, n = 20). Mice in groups C and H were repeatedly inoculated with 0.2 mL 1640 medium containing Coxsackie Virus B3 (CVB3) to build VMC model; at the same time, mice in group H were intraperitoneally injected with 50 mol/kg NaHS; and mice in group H were received intraperitoneal injection of the same amount of normal saline, 1 time/d, for 7 consecutive days. We observed and recorded the symptoms and the survival situation of mice in each group within 7 days, counted the number of live mice at different time points and did analysis about survival rate of each group. We removed dead mice’s hearts after 7 days, weighed them, calculated the HM/BM, observed the pathology change of myocardial cells, calculated myocardial histopathologic score through HE staining, and detected the myocardial apoptosis by TUNEL, and the expression of p53, Bax, Bcl-2, caspase-3 related to apoptotic by Western Blot. Results: Compared with group N, the expression of p53 protein, and Bax protein in group C significantly increased; the expression of caspase-3 protein activity significantly increased; the expression of Bcl-2 protein reduced (p < 0.05). After injecting NaHS to group H, compared with group C, the expression of p53 protein reduced; the expression of Bax protein reduced; the expression of Bcl-2 protein was significantly higher than group C; the expression of caspase-3 protein activity significantly reduced (p < 0.05). Conclusion: These observations suggest that NaHS confers cardioprotection through increasing the survival rate of mice, reducing myocardial pathological scores and myocardial cell apoptosis, and regulating the apoptosis-related proteins p53, Bax, Bcl-2, caspase-3, and inhibition of cardiomyocyte apoptosis effect in acute viral myocarditis.